The calcium (Ca2+)-sensing receptor (CaR or CaSR) "senses" small changes in the extracellular ionized Ca2+ concentration (Ca2+o) and modulates the functions of CaR-expressing cells so as to normalize Ca2+o. Not only parathyroid cells express the CaR but also the thyroidal, calcitonin (CT)-secreting C-cells and cells in several nephron segments within the kidney, as well as various other tissues. Our understanding of the CaR's roles in mineral ion metabolism has been enriched substantially by studying inherited human diseases of the CaR as well as mice with "knockout" (KO) of the receptor (CaR-/-) - initially knockout of only the CaR gene and more recently both the CaR gene and either the Gcm-2 or PTH genes (CaR-/-PTH-/-) to prevent the lethal hyperparathyroidism present in CaR-/- mice. Nevertheless, current understanding of the actions of the CaR in tissues other than the parathyroid is actually quite limited. The preliminary studies for this proposal have revealed striking Ca2+ intake-dependent hypercalcemia in the double KO mice (CaR-/-PTH-/-) compared to control mice (PTH KO, CaR+/+PTH-/-;and wild type, CaR+/+PTH+/+). In addition, there is a nearly complete loss of high Ca2+o-stimulated calcitonin secretion, reduced urinary excretion of Ca2+ at any given level of serum Ca2+, and enhanced sensitivity to exogenous 1,25-dihydroxvitamin D3, which could contribute to the observed phenotypes. The overall goal of this project is to utilize these genetic models as well as mice with conditional KO of the CaR in the kidney to further understand the receptor's involvement in the impaired, PTH-independent ability of the CaR-/-PTH-/- mice to defend against hypercalcemia resulting from a Ca2+ load. The following aims will be addressed: (1) We will characterize the role of the CaR in the kidney in mediating the high Ca2+o-evoked stimulation of urinary Ca2+ excretion as a defense against hypercalcemia. (2) We will investigate the CaR's role in mediating high Ca2+o-induced inhibition of the 1-hydroxylation of 25(OH)D3 as well as intestinal/renal responsiveness to 1,25(OH)2D3 defending against hypercalcemia. (3) We will prove the CaR's role in mediating high Ca2+o-induced stimulation of CT secretion and characterize the importance of loss of CaR-stimulated CT secretion in the hypercalcemia in the c-p- mice from Ca2+ loading. (4) We will study the CaR's role in directly modulating intestinal absorption of Ca2+. These studies will be carried out both in the intact animal to characterize the CaR's role in whole body homeostatic mechanisms as well as in isolated tissues and cells to elucidate the genes contributing to the receptor's in vivo actions. The proposed studies will likely elucidate key, novel Ca2+o-lowering actions of the CaR both in health as well as in various disease states. The goal of this project is to utilize mouse models of human diseases to gain further insight into the mechanisms by which the blood calcium concentration is regulated in health and disease by the extracellular calcium-sensing receptor (CaR), which acts as the body's thermostat for measuring and controlling the calcium concentration in the blood. The full range of mechanisms through which it does so is not well understood, and the studies in this proposal will investigate the importance of the CaR in regulating: (1) the production and action of the body's most active form of vitamin D, (2) the secretion of the calcium-lowering hormone, calcitonin, (3) the absorption of calcium by the intestine, and (4) the manner in which the kidney controls the amount of calcium that is excreted in the urine. These studies should clarify important mechanisms by which the body regulates calcium in bodily fluids under normal circumstances and how these processes can go awry in disease states. PUBLIC HEALTH RELEVAQNCE: The goal of this project is to utilize mouse models of human diseases to gain further insight into the mechanisms by which the blood calcium concentration is regulated in health and disease by the extracellular calcium-sensing receptor (CaR), which acts as the body's thermostat for measuring and controlling the calcium concentration in the blood. The full range of mechanisms through which it does so is not well understood, and the studies in this proposal will investigate the importance of the CaR in regulating: (1) the production and action of the body's most active form of vitamin D, (2) the secretion of the calcium-lowering hormone, calcitonin, (3) the absorption of calcium by the intestine, and (4) the manner in which the kidney controls the amount of calcium that is excreted in the urine. These studies should clarify important mechanisms by which the body regulates calcium in bodily fluids under normal circumstances and how these processes can go awry in disease states.